Method for cutting animal-derived biomembranes

Abstract

The application provides a cutting method of animal-derived biomembrane, and the specific operation process is as follows: biomembrane samples are soaked in a sodium hydroxide solution to make the membrane material swell, then neutralized with an equal volume and concentration of hydrochloric acid solution, and then laid flat on the operating roller of a laser cutting machine with the smooth surface facing downward; during the operation, the roller slowly rotates, the light beam quickly moves left and right, and the part of the fiber layer that is higher than the set height is cut off; or the biomembrane samples are laid flat on the operating platform of a laser cutting machine with the smooth surface facing downward after being taken out, and during the operation, the platform is stationary, the light beam quickly moves left and right and slowly pushes forward, and the part of the fiber layer that is higher than the set height is cut off. After laser cutting during production, the thickness uniformity of the biomembrane intermediate is improved, the cutting yield after freeze-drying is increased, the re-dissolving speed before use is accelerated and stable, and the degradation speed of the biomembrane during use is uniform.

Description

Technical Field

[0001] This invention belongs to the field of regenerative biomedical materials technology and relates to a method for controlling the thickness of animal-derived biofilms during preparation. The method uses ultraviolet laser cutting to control the thickness of animal-derived biofilms. Background Technology

[0002] Existing absorbable membranes are divided into synthetic polymer membranes and animal-derived membranes. Compared to synthetic polymer membranes, animal-derived membranes have a significant drawback: uneven thickness, leading to inconsistent resolution and degradation rates. Current solutions primarily involve physical pressing to reduce the thickness variation of animal-derived membranes. However, this sometimes increases the density variation of the membrane material, making it less effective in addressing the uneven resolution and degradation rates.

[0003] Ultraviolet laser cutting is a new technology with a kerf of 0.1 to 0.3 mm. After special processing, the pericardium thickness is about 1.5 to 5.0 mm. This invention uses ultraviolet laser cutting technology to cut the pericardium, which can make the thickness of the pericardium intermediate product uniform and effectively solve the problem of large thickness difference of animal-derived membranes.

[0004] The pericardium has a natural double-layer structure, with one side smooth and the other loose. The smooth side is the serous membrane layer, which accounts for about 5% of the pericardium's thickness, while the loose side is the fibrous layer, which accounts for about 95% of the pericardium's thickness. Our cutting mainly targets the fibrous layer. When cutting, the smooth side is placed face down and tightly against the roller. Summary of the Invention

[0005] The purpose of this invention is to solve the problem that the physical pressing method used in the prior art sometimes leads to greater density differences in the membrane material, resulting in unsatisfactory effects on solving the problem of uneven resolution and degradation rates.

[0006] To achieve the above objectives, the present invention provides a method for cutting animal-derived biofilms, the specific operation process of which is as follows:

[0007] The biofilm sample is immersed in sodium hydroxide solution to swell the membrane material, then neutralized with an equal volume and concentration of hydrochloric acid solution. After removal, the smooth side is laid flat on the operating roller of the laser cutting machine. During the operation, the roller rotates slowly while the beam moves rapidly left and right to cut off the part of the fiber layer that exceeds the set height. Alternatively, the sample is laid flat on the operating platform of the laser cutting machine with the smooth side facing down. During the operation, the platform remains stationary while the beam moves rapidly left and right and slowly pushes forward to cut off the part of the fiber layer that exceeds the set height.

[0008] Specifically, the following steps are included:

[0009] 1) Take 5 bovine pericardiums, cut and scrape off the appendages on the membranes, and remove the thick areas; then cut each bovine pericardium into 2 pieces to obtain 10 bovine pericardium slices, which are divided into two groups, A and B, as intermediate products.

[0010] 2) Wash the intermediate product with 0.1% to 5.0% sodium chloride solution 3 to 10 times until no blood remains;

[0011] 3) The intermediate product is soaked in 0.1-2.0 mol / L sodium hydroxide solution for 0.5-5 hours to allow the membrane material to swell, and then neutralized with the same volume and concentration of hydrochloric acid solution. At this time, the thickness of the intermediate product is 1.5 mm-5.0 mm.

[0012] Take the intermediate product from group A and cut it with a laser cutting machine to remove the fiber layer portion with a thickness of more than 1.5 to 3.0 mm;

[0013] 4) The intermediate product is washed with 1%–10% hydrogen peroxide working solution for 0.5–6 hours by shaking, and the washing solution is discarded. Then, it is washed 3–10 times with phosphate buffer solution at pH 6.0 and phosphate concentration range of 0.1–0.5 mol / L, each time for 3–30 minutes, to fully neutralize the residual acid and base ions in the intermediate product, and then drained. Finally, it is washed with isopropanol for 1–8 hours for final degreasing, and the isopropanol is discarded.

[0014] 5) Add purified water at a mass ratio of 1:30, and wash by shaking at 100-120 rpm for 5-10 times at room temperature, each time for 10 minutes; finally, spread the intermediate products of group A and group B on plates and freeze-dry them separately.

[0015] 6) After freeze-drying, take samples from both Group A and Group B intermediates to check their thickness. Take samples from each sample to test their tensile strength and conduct degradation experiments under the condition of 200 U / ml type I collagenase.

[0016] The beneficial effects of this invention are: after laser cutting during the production process, the thickness uniformity of the biofilm intermediate is improved, the cutting yield after freeze drying is increased, the reconstitution speed before use is accelerated and stable, and the degradation rate of the biofilm is uniform during use. Detailed Implementation

[0017] To address the issues of uneven thickness (or density) of animal-derived membranes leading to inconsistent reconstitution before use and uneven degradation rates in vivo, this embodiment provides a preferred technical solution: The sample is immersed in a sodium hydroxide solution of a certain concentration to swell the membrane material, then neutralized with an equal volume of hydrochloric acid solution of the same concentration. After removal, the sample is laid flat with the smooth side down on the operating rollers of a laser cutting machine (parameters: wavelength 355nm, power 10W / 15W). During operation, the rollers rotate slowly while the laser beam (torch) moves rapidly left and right, cutting away the portion of the fiber layer exceeding the set height. Alternatively, the sample is immersed in a sodium hydroxide solution of a certain concentration to swell the membrane material, then neutralized with an equal volume of hydrochloric acid solution of the same concentration. After removal, the sample is laid flat with the smooth side down on the operating platform of a laser cutting machine (parameters: wavelength 355nm, power 10W / 15W). During operation, the platform remains stationary while the laser beam (torch) moves rapidly left and right and slowly forward, cutting away the portion of the fiber layer exceeding the set height. Detailed implementation method:

[0019] 1. Take 5 bovine pericardiums, cut and scrape them to remove attached fat, fascia and other tissues, and remove thick areas. Then cut each bovine pericardium into 2 pieces, and divide the 5 bovine pericardiums (10 pieces) into two groups (group A and group B, intermediate products of the two groups).

[0020] 2. Wash the bovine pericardium with 0.1% to 5.0% (optimal concentration range 0.5% to 2.0%) sodium chloride solution 3 to 10 times until the bovine pericardium is free of blood.

[0021] 3. Soak the intermediate product in a sodium hydroxide solution of 0.1–2.0 mol / L (optimal concentration range 0.5–1.0 mol / L) for 0.5–5 hours to allow the membrane material to swell. Then neutralize it with an equal volume of hydrochloric acid solution of the same concentration (the pH value of the solution is measured to be 5.0–7.0). At this time, the sample thickness is 1.5 mm–5.0 mm. Take the intermediate product from group A and cut it with a laser cutter to cut off the fiber layer portion with a thickness exceeding 2.5 mm (the range can be selected as 1.5–3.0 mm).

[0022] 4. The intermediate product is washed with 1%–10% hydrogen peroxide working solution (optimal concentration range 3%–5%) for 0.5–6 hours by shaking, and the washing solution is discarded. Then, it is washed 3–10 times with pH 6.0 phosphate buffer (preferably phosphate concentration range 0.1–0.5 mol / L) for 3–30 minutes each time to fully neutralize the residual acid and base ions in the sample, and drained. Finally, it is washed with isopropanol for 1–8 hours for final degreasing, and the isopropanol is discarded.

[0023] 5. Add purified water to the intermediate product at a material-to-liquid ratio of 1:30 (w / w), and wash with shaking at 100-120 rpm for 5-10 times at room temperature, 10 min / time. Spread the intermediate products of group A and group B onto plates and freeze-dry them separately.

[0024] 6. After freeze-drying, take samples from both groups A and B (10 pieces) at evenly distributed points to check the thickness, and take samples from each piece to test the tensile strength. 注1 Degradation experiments were conducted under the conditions of type I collagenase (200 U / ml).

[0025] Note 1: Tensile strength test

[0026] Take this product and cut the sample into a long strip shape (15mm×5mm). Make a clamping mark 5mm away from the short edge.

[0027] After rehydrating the sample for at least 3 minutes, measure the thickness of the sample in the test area.

[0028] The sample was fixed on the fixture according to the markings on the fixture, and the fixture was pulled at a stable speed of 100 mm / min until the sample broke. The changes in test length and force were continuously monitored throughout the test.

[0029] Calculate the tensile strength (δ) using the following formula:

[0030] δ=F / Wt

[0031] In the formula:

[0032] δ—Tensile strength, in megapascals (MPa);

[0033] F—The corresponding load measured, in Newtons (N);

[0034] W—Width of the sample test area, in millimeters (mm);

[0035] t—Thickness of the sample test area, in millimeters (mm).

[0036] The test results are shown in Tables 1, 2, and 3. The results show that the samples in Group A that underwent laser cutting had smaller deviations in thickness and tensile strength, while the untreated biofilms in Group B showed larger deviations. The degradation test results indicate that thickness deviation directly affects the degradation rate; therefore, using laser cutting as a novel technology to control the thickness of animal-derived biofilms is necessary.

[0037] Table 1:

[0038] The sample thickness inspection results are shown in the table below: Unit: mm

[0039]

[0040] Table 2:

[0041] The tensile strength test results of the samples are shown in the table below:

[0042]

[0043] Table 3:

[0044] The degradation rate test results of type I collagenase degradation experiment are shown in the table below:

[0045] 3h degradation rate 56.66％ 60.18％ 50.38％ 55.52％ 53.36％ 37.09％ 61.67％ 41.54％ 39.32％ 58.51％ 5h degradation rate 83.31％ 87.03％ 79.96％ 82.78％ 81.46％ 69.80％ 87.37％ 72.33％ 70.69％ 83.40％

[0046] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A method for cutting animal-derived biofilms, the specific operation process of which is as follows: The biofilm sample is immersed in sodium hydroxide solution to swell the membrane material, then neutralized with an equal volume and concentration of hydrochloric acid solution. After removal, the smooth side is laid flat on the operating roller of the laser cutting machine. During the operation, the roller rotates slowly while the beam moves rapidly left and right to cut off the part of the fiber layer that exceeds the set height. Alternatively, the sample is laid flat on the operating platform of the laser cutting machine with the smooth side down. During the operation, the platform remains stationary while the beam moves rapidly left and right and slowly pushes forward to cut off the part of the fiber layer that exceeds the set height. The specific steps are as follows: 1) Take 5 bovine pericardiums, cut and scrape off the appendages on the membranes, and remove the thick areas; then cut each bovine pericardium into 2 pieces to obtain 10 bovine pericardium slices, which are divided into two groups, A and B, as intermediate products. 2) Wash the intermediate product with 0.1% to 5.0% sodium chloride solution 3 to 10 times until no blood remains; 3) The intermediate product is soaked in 0.1-2.0 mol / L sodium hydroxide solution for 0.5-5 hours to allow the membrane material to swell, and then neutralized with the same volume and concentration of hydrochloric acid solution. At this time, the thickness of the intermediate product is 1.5 mm-5.0 mm. Take the intermediate product from group A and cut it with a laser cutting machine to remove the fiber layer portion with a thickness of more than 1.5 to 3.0 mm; 4) The intermediate product is washed with 1%–10% hydrogen peroxide working solution by shaking for 0.5–6 hours, and the washing solution is discarded. Then, it is washed 3–10 times with phosphate buffer solution of pH 6.0 and phosphate concentration range of 0.1–0.5 mol / L, each time for 3–30 minutes, to fully neutralize the residual acid and base ions in the intermediate product, and then drained. Finally, it is washed with isopropanol by shaking for 1–8 hours for final degreasing, and the isopropanol is discarded. 5) Add purified water at a mass ratio of 1:30, and wash by shaking at 100-120 rpm for 5-10 times at room temperature, each time for 10 minutes; finally, spread the intermediate products of group A and group B on plates and freeze-dry them separately. 6) After freeze-drying, take samples from both Group A and Group B intermediates to check their thickness, test the tensile strength of each sample, and conduct a degradation experiment under the condition of 200 U / ml type I collagenase.

2. The method for cutting animal-derived biofilms according to claim 1, characterized in that, The concentration of the sodium chloride solution is 0.5%–2.0%, and the concentration of the sodium hydroxide solution is 0.5–1.0 mol / L; the pH value of the neutralized intermediate is 5.0–7.0; the concentration of the hydrogen peroxide working solution is 3%–5%; and the concentration of the phosphate buffer solution is 0.1–0.5 mol / L.

3. The method for cutting animal-derived biofilms according to claim 1 or 2, characterized in that, The accessory tissues refer to fat or fascia.

4. The method for cutting animal-derived biofilms according to claim 1 or 2, characterized in that, The specific method for sampling and testing the tensile strength of each piece is as follows. Take this product and cut the sample into a strip shape of 15mm×5mm. Make a clamp to hold the mark 5mm away from the edge of the short side. After rehydrating the sample for at least 3 minutes, measure the thickness of the sample in the test area. The sample was fixed on the fixture according to the markings, and the fixture was pulled at a steady speed of 100 mm / min until the sample broke. Throughout the test, the changes in test length and force were continuously monitored, and the tensile strength (δ) was calculated using the following formula: δ=F / Wt, where: δ—tensile strength, in megapascals (MPa); F—the corresponding load measured, in newtons (N); W—width of the sample test area, in millimeters (mm); t—thickness of the sample test area, in millimeters (mm).

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